Use of plastid transit peptides derived from glaucocystophytes

ABSTRACT

Compositions and methods for targeting polypeptides to plastids are provided. Compositions comprise plastid transit peptides as well as nucleotide sequences encoding such transit peptides and variants thereof. Compositions further comprise DNA constructs comprising a nucleotide sequence encoding the plastid transit peptide operably linked to a nucleotide sequence encoding a polypeptide of interest. These DNA constructs find use in expression and targeting of the polypeptide of interest to a plastid. Compositions also comprise expression cassettes, vectors, transformed plants, transformed plant cells, and stably transformed plant seeds wherein a polypeptide of interest is targeted to a plastid by the plastid transit peptide of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/024,844, filed Jan. 30, 2008. This application also claims priorityfrom U.S. Provisional Application No. 61/047,692, filed Apr. 24, 2008.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of “Sequence Listing 71999.txt”, a creation date of Jan. 10, 2009,and a size of 7.25 KB. The sequence listing filed via EFS-Web is part ofthe specification and is hereby incorporated in its entirety byreference herein.

FIELD OF THE INVENTION

The present invention relates to the genetic modification of plants,particularly to the transport of polypeptides of interest to plantplastids.

BACKGROUND OF THE INVENTION

The plastid contains it own genome. However, during evolution many ofthe plastid genes were transferred to the nuclear genome. Thus, amechanism for the transport of the nuclear encoded plastid proteins backto the plastid was developed. The proteins are transported through theuse of transit peptide sequences located in the N-terminus of thetransported proteins. These peptides direct the proteins to the plastidand can be subsequently recognized by specific proteases which can leadto removal of the transit peptide.

A fundamental problem in cell biology is the precise and efficienttargeting of proteins synthesized by cytoplasmic ribosomes to theirappropriate intracellular locations. This is especially true fortransgenic higher plants where the transgene product is needed in anappropriate cellular organelle. The present invention provides plastidtransit peptides that efficiently transport a heterologous polypeptideinto the chloroplast of transgenic higher plants. Surprisingly, this canbe accomplished using a cyanelle transit peptide derived from aGlaucocystophyte.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for targeting polypeptides to chloroplasts areprovided. Compositions comprise transit peptides as well as nucleotidesequences encoding a transit peptide (i.e., plastid targeting peptide ortransport peptide) and variants thereof. Compositions further compriseDNA constructs comprising a nucleotide sequence encoding the transitpeptide operably linked to a nucleotide sequence encoding a heterologouspolypeptide. These DNA constructs find use in expression and targetingof the heterologous polypeptide to a plastid. Compositions also compriseexpression cassettes, vectors, transformed plants, transformed plantcells, and stably transformed plant seeds comprising a polynucleotideencoding a heterologous polypeptide which is targeted to a chloroplastby the plastid transit peptide of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The plastid targeting or transport peptides of the invention mediatetargeting, localization, or transport of operably linked polypeptidesinto plastids. Plastids are organelles found in plants and algae.Plastids are responsible for photosynthesis, storage of products such asstarch, and for the synthesis of many classes of molecules such as fattyacids, terpenes, and other molecules which are needed as cellularbuilding blocks. Plastids have the ability to differentiate, orredifferentiate, into several forms depending upon their role in thecell. Undifferentiated proplastids may develop into any of the followingplastids, including, chloroplasts, chromoplasts, leucoplasts,amyloplasts, statoliths, elaioplasts, and proteinoplasts.

A “chloroplast transit peptide” or “plastid transport peptide” or“plastid transit peptide” or “plastid targeting peptide” or “transitpeptide” or “targeting peptide” is necessary and sufficient tofacilitate the import of a protein into the plastid of its native hostcell. The plastid may be a cyanelle or a chloroplast. Transit peptidesare located at the N-terminal end of the proteins imported into theplastids. The transit peptide facilitates co-translational orpost-translational transport of an operably linked polypeptide into aplastid.

These transit peptides generally comprise between 40 and 100 aminoacids. Studies indicate that transit peptides contain commoncharacteristics. These include: they are virtually devoid of negativelycharged amino acids, such as aspartic acid, glutamic acid, asparagine orglutamine; the N-terminal region is devoid of charged amino acids, andof amino acids such as glycine or proline; their central region containsa very high proportion of basic or hydroxylated amino acids, such asserine or threonine; and, their C-terminal region is rich in arginineand has the ability to form an amphipathic beta-sheet secondarystructure.

According to one authority (Cline and Henry, Annu Rev Cell Dev Biol 12:1-26 (1996)) chloroplast transit peptides from higher plants share thefollowing characteristics: (1) They have superficially similarproperties to mitochondrial transit peptides. That is they are rich inhydroxylated residues and poor in acidic residues. (2) They are 30-120residues long. (3) The N-terminal 10-15 amino acids are devoid ofglycine, proline and charged residues. (4) The variable, middle regionis rich in serine, threonine, lysine and arginine. (5) The C-proximalregion contains the loosely conserved sequence (Ile/Val-x-Ala/Cys*Ala)for proteolytic processing. (6) There is no extended sequenceconservation or conserved secondary structural motifs (7) They,theoretically, adopt a predominantly random coil conformation.

Several computational approaches exist which use the above features topredict chloroplast targeting sequences from higher plants.Computational tools include PSORT (Nakai and Kanehisa, Proteins 11(2):95-110 (1991); Horton et al., Proceedings of the 4^(th) Annual AsiaPacific Bioinformatics Conference APBC06, Taipei, Taiwan pp. 39-48(2006); http://www.psort.org), ChloroP (Emanuelsson et al., J Mol Biol300: 1005-1016 (1999); http://www.cbs.dtu.dk/services/ChloroP/) orTargetP (Nielsen et al., Protein Engineer 10:1-6 (1997); Emanuelsson etal., J Mol Biol 300: 1005-1016 (2000);http://www.cbs.dtu.dk/services/TargetP/).

Glaucocystophytes are a family of algae that contain cyanelles as aphotosynthetic organelle. Several genera exist within theGlaucocystophytes including Cyanophora, Glaucocystis and Gloeochaete.Cyanelles are in essence primitive plastids. Unlike other eukaryoticplastids, cyanelles have a peptidoglycan layer which is believed to be arelic of the endosymbiotic origin of plastids from cyanobacteria.Glaucocystophytes contain the photosynthetic pigment chlorophyll a.Along with red algae and cyanobacteria they harvest light viaphycobilisomes, structures consisting largely of phycobiliproteins. Thegreen algae and land plants have lost that pigment.

Amongst all of the platids, Glaucocystophyte cyanelles are the closestrelatives to the free-living cyanobacteria. The presence of thepeptidioglycan layer in the cell wall of the cyanelles is likely to be akey component of the protein import machinery of the cyanelle. SeeSteiner and Loffelhardt, Molecular Membrane Biology 22(1-2): 123-132(2005) for a review of cyanelle protein translocation. The chloroplastsof higher plants have lost the peptidioglycan layer and thus differ intheir membrane structure from cyanelles. It was further demonstratedthat the import machinery of cyanelles and higher plants differ as thecyanelle targeted protein, ferredoxin-NADP⁺-oxidoreductase, was importedinto isolated pea and spinach chloroplasts (Steiner and Loffelhardt,Trends Plant Sci 7: 72-77 (2002)); however, higher plant chloroplasttargeted proteins were not functional with cyanelles (Steiner andLoffelhardt Mol. Memb. Biol. 22:123-132 (2005)).

Cyanelle transit peptide sequences differ from chloroplast stromatargeting peptides in containing in their N-terminal domain an invariantphenylalanine residue which is crucial for import by the cyanelle. See,for example, Steiner and Loffelhardt Trends Plant Sci. 7:72-77 (2002);and Steiner and Loffelhardt Mol. Memb. Biol. 22:123-132 (2005). Theinvariant phenylalanine residue is unique to cyanelles and therhodoplast (red algae plastids) and is not a feature of the plastidtransit peptide in the green lineage organisms (green algae, Euglena,diatoms and higher plants). The cyanelle transit sequences correspond tostroma-targeting peptides from higher plants in their amino acidcomposition, in their positive net charge and in their domain structure.The N-termini of cyanelle transit peptides show a short consensussequence. See, Steiner et al. (2005) The Plant J. 44:646-652 (2005).While not bound to any mechanism of action, it is believed that theN-terminal phenylalanine residue is only necessary for importation intocyanelles, thus, this residue may be modified in the practice of thepresent invention and still effectively transport a polypeptide into ahigher plant chloroplast.

Taken together, the above data raises doubts on the ability of cyanelletargeting sequences to direct heterologous proteins to the chloroplastsof higher plants. While the Cyanophora paradoxa pre-FNR molecule wasable to enter isolated chloroplasts from pea, it does not necessarilyfollow that the FNR transit peptide when attached to a heterologousprotein would be able to interact with the higher plant secretory systemto effectively target a heterologous protein to the chloroplast.Surprisingly, the instant application describes methods for targetingheterologous proteins to chloroplasts in higher plants using cyanelletransit peptides.

In one embodiment of the invention, the transit peptide is from aGlaucocystophyte which when operably linked to a heterologous proteinand expressed in a transgenic higher plant, targets the heterologousprotein to the chloroplast. The Glaucocystophyte is selected from thegroup consisting of Cyanophora, Glaucocystis, Gloeochaete and Cyanophoraparadoxa.

Another embodiment of the invention is a method of targeting aheterologous protein to the chloroplast of a transgenic higher plantcomprising the steps of operably linking a transit peptide from aGlaucocystophyte to a heterologous protein and generating a transgenicplant comprising the heterologous protein wherein the heterologousprotein is detected in the chloroplast of the transgenic plant. TheGlaucocystophyte is selected from the group consisting of Cyanophora,Glaucocystis, Gloeochaete and Cyanophora paradoxa.

The compositions comprise nucleotide sequences encoding a transitpeptide as well as variants thereof. In one embodiment the transitpeptide comprises the amino acid sequence set forth in SEQ ID NO:2 orfragments and variants thereof as well as the nucleotide sequence setforth in SEQ ID NO:1 and variants thereof. Compositions further compriseDNA constructs comprising a nucleotide sequence encoding the transitpeptide operably linked to a nucleotide sequence encoding a heterologouspolypeptide. These DNA constructs find use in expression and targetingof the heterologous polypeptide to a plastid. Compositions also compriseexpression cassettes, vectors, transformed plants, transformed plantcells, and stably transformed plant seeds wherein a heterologouspolypeptide is targeted to a plastid by the transit peptide of theinvention.

In another embodiment of the invention, the transit peptide is from aGlaucocystophyte which when operably linked to a heterologous proteinand expressed in a transgenic higher plant, targets the heterologousprotein to the chloroplast. Transit peptides are derived from thenucleic acid sequence encoding a protein which is targeted to thecyanelle of the Glaucocystophyte. Transit peptides can be found in genesknown to be encoded in the nucleus of the host cell but upon translationto be targeted to the photosynthetic organelle of the host cell and canbe selected from the group consisting of ribulose bisphosphatecarboxylase/oxygenase, 5-enolpyruvyl-shikimate-3-phosphate synthase,acetolactate synthase, chloroplast ribosomal protein CS17, Cs protein,ferredoxin, plastocyanin, ribulose bisphosphate carboxylase activase,tryptophan synthase, acyl carrier protein, plastid chaperonin-60,cyochrome C552, 22-kDA heat shock protein, 33-kDA oxygen-evolvingenhancer protein 1, ATP synthase gamma subunit, ATP synthase omegasubunit, chlorophyll-a/b-binding proteinII-1, oxygen-evolving enhancerprotein 2, oxygen-evolving enhancer protein 3, photosystem I P21,photosystem I P28, photosystem I P30, photosystem I P35, photosystem IP37, glycerol-3-phosphate acyltransferases, chlorophyll a/b bindingprotein, CAB2 protein, hydroxymethyl-bilane synthase,pyruvate-orthophosphate dikinase, CAB3 protein, plastid ferritin,ferritin, early light-inducible protein, glutamate-1-semialdehydeaminotransferase, protochlorophyllide reductase, starch-granule-boundamylase synthase, light-harvesting chlorophyll a/b-binding protein ofphotosystem II, major pollen allergen Lol p 5a, plastid ClpB ATPdependent protease, superoxide dismutase, ferredoxin NADPoxidoreductase, 28-kDa ribonucleotoprotein, 31-kDa ribonucleoprotein,33-kDa ribonucleoprotein, acetolacate synthase, ATP synthase CFO subunit1, 2, 3, or 4; cytochrome f, ADP-glucose pyrophosphorylase, glutaminesynthase, glutamine synthase 2, carbonic anhydrase, GapA protein, heatshock protein hsp 21, phosphate translocator, plastid CIpA ATP dependentprotease, plastid ribosomal protein CL24, plastid ribosomal protein CL9,plastid ribosomal protein PsCL18, plastid ribosomal protein PsCL25, DAHPsynthase, starch phosphorylase, root acyl carrier protein 11,betaine-aldehyde dehydrogase, GapB protein, glutamine synthase 2,phosphoribulokinase, nitrite reductase, ribosomal protein L12, ribosomalprotein L13, ribosomal protein L21, ribosomal protein L35, ribosomalprotein L40, triose phosphate-3-phosphoglyerate phosphate translocator,ferredoxin dependent glutamate synthase, glyceraldehyde 3 phosphatedehydrogenase, NADP dependent malic enzyme and NADP malatedehydrogenase. Table 1 identifies additional nuclear encoded genescontaining plastid targeting sequences. In one embodiment of theinvention the transit peptide is from Cyanophora pardoxa, particularlyfrom C. pardoxa ferredoxin-NADP⁺-oxidoreductase and comprises the aminoacid sequence set forth in SEQ ID NO:2.

TABLE 1 Nuclear genes encoding proteins targeted to the plastid.References are herein incorporated by reference in their entirety.Transit Reference peptides Organism Steiner and Loffelhardt, FIG. 2,Cyanophora paradoxa Trends in Plant Science 7(2): page 74 72-77 (2002)Reyes-Prieto and Table 1, Calvin Cycle genes Bhattacharya, Molecularpage 385 which function in the Phylogentics and Evolution chloroplastbut are 45: 384-391 (2007) encoded in the nucleus Schwartzbach et alPlant FIG. 1, Cyanophora paradoxa Molecular Biology 38: 247- page 249263 (1998) Steiner et al, The Plant FIG. 1, Cyanophora paradoxa Journal44: 646-652 (2005) page 647

The transit peptides of the invention include variant or modifiedsequences. Such sequences have altered amino acid sequences yet retainthe ability to transport a linked polypeptide into a plastid. Whilemethods are known for substituting amino acid residues in polypeptidesequences, it is recognized that serine and threonine have beenidentified as abundant in transit peptides in comparison to the entirechloroplast targeting protein. Aspartic acid and glutamic acid have beenreported as under represented in transit peptides. Such considerationsas well as those discussed above may be taken into account whenconstructing variant transit peptides. However, as detailed below, oneof skill can assay whether a variant transit peptide is capable ofeffecting transport of an operably linked polypeptide by assaying forthe presence of the operably linked polypeptide in the plastid.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

By a “crop plant” or “higher plant” is intended any plant that iscultivated for the purpose of producing plant material that is soughtafter by man for either oral consumption, or for utilization in anindustrial, pharmaceutical, or commercial process. The invention may beapplied to any of a variety of plants, including, but not limited tomaize, wheat, rice, barley, soybean, cotton, sorghum, oats, tobacco,Miscanthus grass, Switch grass, trees, beans in general, rape/canola,alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet,cocoa, tea, Brassica, cotton, coffee, sweet potato, flax, peanut,clover; vegetables such as lettuce, tomato, cucurbits, cassava, potato,carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brusselssprouts, peppers, and pineapple; tree fruits such as citrus, apples,pears, peaches, apricots, walnuts, avocado, banana, nut and coconut; andflowers such as orchids, carnations, roses, and the like.

As used herein, the term “plant part” or “plant tissue” includes plantcells, plant protoplasts, plant cell tissue cultures from which plantscan be regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants such as embryos, pollen, ovules,seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks,stalks, roots, root tips, anthers, and the like.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element. Throughout thespecification the word “comprising,” or variations such as “comprises”or “comprising,” will be understood to imply the inclusion of a statedelement, integer or step, or group of elements, integers or steps, butnot the exclusion of any other element, integer or step, or group ofelements, integers or steps.

Transit Peptide Compositions

As indicated, the compositions of the invention include transit peptidescapable of transporting operably linked polypeptides into plastids. Theterm “peptide” broadly refers to an amino acid chain that includesnaturally occurring amino acids, synthetic amino acids, geneticallyencoded amino acids, non-genetically encoded amino acids, andcombinations thereof. Peptides can include both L-form and D-form aminoacids.

Representative non-genetically encoded amino acids include but are notlimited to 2-aminoadipic acid; 3-aminoadipic acid; β-aminopropionicacid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid);6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid;3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid;desmosine; 2,2′-diaminopimelic acid; 2,3-diaminoproprionic acid;N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine;3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine;N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline;norvaline; norleucine; and ornithine.

Representative derivatized amino acids include, for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups can be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups canbe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine can be derivatized to form N-im-benzylhistidine.

The plastid transit peptide is generally fused N-terminal to thepolypeptide to be transported into a plastid. It is recognized thatadditional amino acid residues may be N-terminal to the plastid transitpeptide. The plastid transit peptide is generally cleaved from thepolypeptide of interest upon localization to the plastid. The positionof cleavage may vary slightly between plant species.

The transit peptides of the invention include the transit peptide from aCyanophora ferredoxin-NADP⁺-oxidoreductase, particularly the transitpeptide from a Cyanophora pardoxa ferredoxin-NADP⁺-oxidoreductase, moreparticularly the amino acid sequence set forth in SEQ ID NO:2.Biologically active variants of the peptides of the invention are alsoencompassed by the present invention. Such variants should retain theability to transport an operatively linked polypeptide into a cellularplastid. Preferably, the variant has at least the same activity as thenative molecule. The ability to transport polypeptides into a plastidcan be measured by methods in the art. For example, the nucleotidesequence encoding the transit peptide can be linked to a reporter genesuch as chloramphenicol acetyl transferase (CAT) and β-glucuronidase(GUS) and introduced into a plant. Analysis of CAT and GUS activities inthe subcellular fractions of the transformed plant indicate whether thetransit peptide is capable of targeting the reporter proteins to theplastids. See, for example, Silva-Filho et al. (1997) J Biol Chem272:15264-15269 and US Patent Application No. 20080168580.

Suitable biologically active variants can be fragments and derivatives.By “fragment” is intended a peptide consisting of only a part of theintact transit peptide sequence and structure, and can be a C-terminaldeletion or N-terminal deletion of amino acids or deletions at both theC- and N-terminal ends. By “derivatives” is intended any suitablemodification of a transit peptide or peptide fragment encompassing anychange in amino acid residues, so long as the transport activity isretained. For example, without being limited, the invariantphenylalanine residue on the cyanelle transit peptide can be used toidentify cyanelle transit peptide sequences; however, this residue maybe changed to another amino acid to create a derivative that is alsofunctional in transgenic higher plants to target heterologous proteinsto chloroplasts.

Peptide variants will generally have at least 70%, preferably at least80%, more preferably about 90% to 95% or more, about 96%, about 97%, andmost preferably about 98%, about 99% or more amino acid sequenceidentity to the amino acid sequence of the reference peptide molecule. Avariant may differ by as few as 5, 4, 3, 2, or even 1 amino acidresidue. Methods for determining identity between sequences are wellknown in the art. See, for example, the ALIGN program (Dayhoff (1978) inAtlas of Protein Sequence and Structure 5:Suppl. 3 (National BiomedicalResearch Foundation, Washington, D.C.) and programs in the WisconsinSequence Analysis Package, Version 8 (available from Genetics ComputerGroup, Madison, Wis.), for example, the GAP program. For purposes ofoptimal alignment of the two sequences, the contiguous segment of theamino acid sequence of the variant may have additional amino acidresidues or deleted amino acid residues with respect to the amino acidsequence of the reference molecule. The contiguous segment used forcomparison to the reference amino acid sequence will comprise at leasttwenty (20) contiguous nucleotides, and may be about 3, about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 15, about 20, about 25, about 30, about 40 or more nucleotides.Corrections for increased sequence identity associated with inclusion ofgaps in the variant's amino acid sequence can be made by assigning gappenalties. Methods of sequence alignment are well known in the art.

When considering percentage of amino acid sequence identity, some aminoacid residue positions may differ as a result of conservative amino acidsubstitutions, which do not affect properties of protein function. Inthese instances, percent sequence identity may be adjusted upwards toaccount for the similarity in conservatively substituted amino acids.Such adjustments are well known in the art. See, for example, Meyers andMiller (1988) Computer Applic. Biol. Sci. 4:11-17.

For example, preferably, conservative amino acid substitutions may bemade. A “nonessential” amino acid residue is a residue that can bealtered without altering the biological activity, whereas an “essential”amino acid residue is required for biological activity. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). See, forexample, Sambrook J., and Russell, D. W. (2001) Molecular Cloning. ALaboratory Manual. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methodsand Applications (Academic Press, NY).

The peptides of the invention can be subject to various changes,substitutions, insertions, and deletions where such changes provide forcertain advantages in its use. Thus, the term “peptide” encompasses anyof a variety of forms of peptide derivatives including, for example,amides, conjugates with proteins, cyclone peptides, polymerizedpeptides, conservatively substituted variants, analogs, fragments,chemically modified peptides, and peptide mimetics. Any peptide that hasdesired transport characteristics can be used in the practice of thepresent invention.

By “transports,” “targets,” or “transfers” is intended that the transitpeptides of the invention are capable of transporting, transferring orcarrying polypeptides expressed in the nucleus of the cell into acellular plastid, for example, without limitation, a chloroplast. Insome embodiments, the transit peptides of the invention are capable oftransporting 100%, at least 90%, at least 80%, at least 70%, at least60%, at least 50% of an operably linked polypeptide into a plastid.

“Isolated” means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living animal inits natural state is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. For example, with respectto polynucleotides, the term isolated means that it is separated fromthe chromosome and cell in which it naturally occurs. A sequence is alsoisolated if separated from the chromosome and cell in which it naturallyoccurs in but inserted into a genetic context, chromosome, or cell inwhich it does not naturally occur.

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding transit peptides orbiologically active portions thereof. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

Nucleic acid molecules that are fragments of these transit peptideencoding nucleotide sequences are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a transit peptide. Nucleic acid molecules that arefragments of a transit peptide nucleotide sequence comprise at leastabout 15, about 20, about 50, about 75, about 100, about 125, about 150,about 175, about 180, about 185, about 190, about 195 contiguousnucleotides. By “contiguous” nucleotides is intended nucleotide residuesthat are immediately adjacent to one another.

The skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedtransit peptides, without altering the transport. Thus, variant isolatednucleic acid molecules can be created by introducing one or morenucleotide substitutions, additions, or deletions into the correspondingnucleotide sequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedpeptide. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Such variantnucleotide sequences are also encompassed by the present invention.

The compositions of the invention can be used to identify and isolatecorresponding transit peptides from other organisms. In one method, thesequence can be used in hybridization assays to find sequences withsubstantial homology to the sequences of the invention. As used herein,the term “hybridization” is used in reference to the pairing ofcomplementary (including partially complementary as discussed above)polynucleotide strands. Hybridization and the strength of hybridization(i.e., the strength of the association between polynucleotide strands)is impacted by many factors well known in the art including the degreeof complementarity between the polynucleotides; stringency of theconditions involved that are affected by such conditions as theconcentration of salts; the melting temperature (Tm) of the formedhybrid; the presence of other components; the molarity of thehybridizing strands; and, the G:C content of the polynucleotide strands.

Thus, isolated sequences that are capable of transporting polypeptidesinto a plastid and which hybridize under stringent conditions to thesequences disclosed herein, or to fragments thereof, are encompassed bythe present invention. Such sequences will be at least about 40% to 50%homologous, about 60%, 65%, or 70% homologous, and even at least about75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or morehomologous with the disclosed sequence[s]. That is, the sequenceidentity of sequences may range, sharing at least about 40% to 50%,about 60%, 65%, or 70%, and even at least about 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

As used herein, “Tm” and “melting temperature” are interchangeable termswhich are the temperature at which 50% of a population ofdouble-stranded polynucleotide molecules becomes dissociated into singlestrands. The equation for calculating the Tm of polynucleotides is wellknown in the art. For example, the Tm may be calculated by the followingequation: Tm=69.3+0.41×(G+C) %−650/L, wherein L is the length of theprobe in nucleotides. The Tm of a hybrid polynucleotide may also beestimated using a formula adopted from hybridization assays in 1 M salt,and commonly used for calculating Tm for PCR primers: [(number ofA+T)×2° C.+(number of G+C)×4° C.], see, for example, Newton et al.(1997) PCR 2nd Ed. (Springer-Verlag, New York). Other more sophisticatedcomputations exist in the art, which take structural as well as sequencecharacteristics into account for the calculation of Tm. A calculated Tmis merely an estimate; the optimum temperature is commonly determinedempirically.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Corresponding sequences can also be identified by PCR reactions. See,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). See alsoInnis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). The primers of thepresent invention can be prepared using techniques known in the art,including, but not limited to, cloning and digestion of the appropriatesequences and direct chemical synthesis.

Chemical synthesis methods that can be used to make the primers of thepresent invention, include, but are not limited to, the phosphotriestermethod described by Narang et al., Methods in Enzymology, 68:90 (1979),the phosphodiester method disclosed by Brown et al., Methods inEnzymology, 68:109 (1979), the diethylphosphoramidate method disclosedby Beaucage et al., Tetrahedron Letters, 22:1859 (1981) and the solidsupport method described in U.S. Pat. No. 4,458,066. The use of anautomated oligonucleotide synthesizer to prepare syntheticoligonucleotide primers of the present invention is also contemplatedherein. Additionally, if desired, the primers can be labeled usingtechniques known in the art and described below.

Plant Expression Cassettes

As discussed, the plastid transit peptides of the invention operablylinked to a polypeptide of interest transports the linked polypeptideinto a plastid. Thus, when assembled into a DNA construct and used totransform a plant, the transit peptide directs a polypeptide of interestto plastids in the transformed plant cells, plants, and seeds afterexpression. Thus, the compositions of the invention also comprisenucleic acid sequences for transformation and expression andaccumulation of a polypeptide of interest in the plastids of a plantcell of interest. The nucleic acid sequences may be present in DNAconstructs or expression cassettes. “Expression cassette” as used hereinmeans a nucleic acid molecule capable of directing expression of aparticular nucleotide sequence in an appropriate host cell, comprising apromoter operatively linked to the nucleotide sequence of interest(i.e., a nucleotide sequence encoding a polypeptide of interest) whichis operatively linked to termination signals. It also typicallycomprises sequences required for proper translation of the nucleotidesequence. The expression cassette comprising the nucleotide sequence ofinterest may be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.Typically, however, the expression cassette is heterologous with respectto the host, i.e., the particular DNA sequence of the expressioncassette does not occur naturally in the host cell and must have beenintroduced into the host cell or an ancestor of the host cell by atransformation event. The expression of the nucleotide sequence in theexpression cassette may be under the control of a constitutive promoteror of an inducible promoter that initiates transcription only when thehost cell is exposed to some particular external stimulus. Additionally,the promoter can also be specific to a particular tissue or organ orstage of development.

The present invention encompasses the transformation of plants withexpression cassettes capable of directing expression and accumulation ofa polypeptide of interest in the plastids of a plant cell. Theexpression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a polynucleotide encoding a plastid transit peptide,and a polynucleotide encoding a polypeptide of interest. The expressioncassette may optionally comprise a transcriptional and translationaltermination region (i.e. termination region) functional in plants.

In addition to the polynucleotide sequence encoding the plastid transitpeptide, the construct may further comprise additional regulatoryelements to facilitate transcription, translation, or transport of thepolypeptide of interest. The regulatory sequences of the expressionconstruct are operably linked to the polynucleotide of interest. By“operably linked” is intended a functional linkage between a regulatoryelement and a second sequence wherein the regulatory element initiatesand/or mediates transcription, translation, or translocation of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleotide sequences being linked are contiguous.The regulatory elements include promoters, enhancers, and signalsequences useful for targeting cytoplasmically-synthesized proteins toplastids of the plant cell. In one embodiment, the construct comprises,in the 5′ to 3′ direction of transcription, a transcriptional andtranslational initiation region (i.e., a promoter), a polynucleotideencoding a plastid transit peptide, and a polynucleotide encoding apolypeptide of interest.

Any promoter capable of driving expression in the plant of interest maybe used in the practice of the invention. The promoter may be native oranalogous or foreign or heterologous to the plant host. The terms“heterologous” and “exogenous” when used herein to refer to a nucleicacid sequence (e.g. a DNA or RNA sequence) or a gene, refer to asequence that originates from a source foreign to the particular hostcell or, if from the same source, is modified from its original form.Thus, a heterologous gene in a host cell includes a gene that isendogenous to the particular host cell but has been modified through,for example, the use of DNA shuffling. The terms also includenon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid (e.g.DNA or RNA) sequence naturally associated with a host cell into which itis introduced.

The choice of promoters to be included depends upon several factors,including, but not limited to, efficiency, selectability, inducibility,desired expression level, and cell- or tissue-preferential expression.It is a routine matter for one of skill in the art to modulate theexpression of a sequence by appropriately selecting and positioningpromoters and other regulatory regions relative to that sequence.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. Thus, as used herein a cell type- ortissue-preferential promoter is one that drives expressionpreferentially in the target tissue, but may also lead to someexpression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos, et al., PlantCell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044 (1988);Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., PlantPhysiology 110: 1069-1079 (1996).

Promoters active in photosynthetic tissue in order to drivetranscription in green tissues such as leaves and stems are ofparticular interest for the present invention. Most suitable arepromoters that drive expression only or predominantly in such tissues.The promoter may confer expression constitutively throughout the plant,or differentially with respect to the green tissues, or differentiallywith respect to the developmental stage of the green tissue in whichexpression occurs, or in response to external stimuli.

Examples of such promoters include the ribulose-1,5-bisphosphatecarboxylase (RbcS) promoters such as the RbcS promoter from easternlarch (Larix laricina), the pine cab6 promoter (Yamamoto et al. (1994)Plant Cell Physiol. 35:773-778), the Cab-1 gene promoter from wheat(Fejes et al. (1990) Plant Mol. Biol. 15:921-932), the CAB-1 promoterfrom spinach (Lubberstedt et al. (1994) Plant Physiol. 104:997-1006),the cab1R promoter from rice (Luan et al. (1992) Plant Cell 4:971-981),the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuokaet al. (1993) Proc Natl Acad Sci USA 90:9586-9590), the tobacco Lhcb1*2promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), theArabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al.(1995) Planta 196:564-570), and thylakoid membrane protein promotersfrom spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Otherpromoters that drive transcription in stems, leafs and green tissue aredescribed in U.S. Patent Publication No. 2007/0006346, hereinincorporated by reference in its entirety.

A maize gene encoding phosphoenol carboxylase (PEPC) has been describedby Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Usingstandard molecular biological techniques the promoter for this gene canbe used to drive the expression of any gene in a green tissue-specificmanner in transgenic plants.

In some other embodiments of the present invention, inducible promotersmay be desired. Inducible promoters drive transcription in response toexternal stimuli such as chemical agents or environmental stimuli. Forexample, inducible promoters can confer transcription in response tohormones such as giberellic acid or ethylene, or in response to light ordrought.

A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and correct mRNA polyadenylation. Thetermination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Appropriatetranscriptional terminators are those that are known to function inplants and include the CAMV 35S terminator, the tml terminator, thenopaline synthase terminator and the pea rbcs E9 terminator. These canbe used in both monocotyledons and dicotyledons. In addition, a gene'snative transcription terminator may be used.

In some embodiments, the expression cassette will comprise a selectablemarker gene for the selection of transformed cells. Selectable markergenes are utilized for the selection of transformed cells or tissues.

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adhl gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronze 1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).Other leader sequences known in the art include but are not limited to:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS USA86:6126-6130 (1989)); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader, (Macejak, D. G., and Samow, P., Nature353: 90-94 (1991); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV), (Gallie,D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and MaizeChlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology84:965-968 (1987).

As indicated the plastid transit peptide is operably linked to apolypeptide to be transported into the plastid. Polypeptides for traitsof interest include agronomic traits that primarily are of benefit to aseed company, a grower, or a grain processor, for example, herbicideresistance, virus resistance, bacterial pathogen resistance, insectresistance, nematode resistance, and fungal resistance. See, e.g., U.S.Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431.Traits of interest may also be one that increases plant vigor or yield(including traits that allow a plant to grow at different temperatures,soil conditions and levels of sunlight and precipitation), or one thatallows identification of a plant exhibiting a trait of interest (e.g.,selectable marker gene, seed coat color, etc.). A plethora of genesuseful for generating plants with desired secondary traits are availablein the art.

Herbicide resistance genes of interest include those encoding precursorsof acetolactase synthetase (ALS), see, for example, U.S. Pat. No.5,013,659; mutated acetolactate synthetase;3-enolpyruvylshikimate-5-phosphate synthetase (EPSP synthetase), see,for example, U.S. Pat. Nos. 4,971,908 and 6,225,114; enzymes that modifya physiological process that occurs in a plastid includingphotosynthesis, fatty acid, amino acid, oil, arotenoid, terpenoid, andstarch; etc. Other genes of interest include but are not limited tothose encoding zeaxanthin epoxidase, choline monooxygenase,ferrochelatase, omega-3 fatty acid desaturase, glutamine synthetase,starch modifying enzymes, essential amino acids, provitamin A, hormones,Bt toxin proteins, and the like. Nucleotide sequences for suchpolypeptides are available in the art.

Plants

Plants useful in the present invention include plants that aretransgenic for at least a polynucleotide encoding a plastid-targetedpolypeptide of interest. Such plants will comprise a polynucleotideencoding a transit peptide operably linked to a polynucleotide encodinga polypeptide of interest. One of skill in the art will recognize thatthe polypeptide sequences of interest may be associated with orcontributing to one or more secondary trait(s) of interest. Thesepolypeptides are cytoplasmically-expressed, and targeted to a plastid.

The type of plant selected depends on a variety of factors, includingfor example, the downstream use of the harvested plant material,amenability of the plant species to transformation, and the conditionsunder which the plants will be grown, harvested, and/or processed. Oneof skill will further recognize that additional factors for selectingappropriate plant varieties for use in the present invention includehigh yield potential, good stalk strength, resistance to specificdiseases, drought tolerance, rapid dry down and grain quality sufficientto allow storage and shipment to market with minimum loss.

It is further contemplated that the constructs of the invention may beintroduced into plant varieties having improved properties suitable oroptimal for a particular downstream use. Plants useful in the presentinvention include, but are not limited to, monocotyledonous anddicotyledonous plants, particularly crop plants such as maize, soybean,wheat, rice, Brassica, and the like. Examples of plant species ofinterest include, but are not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereals), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Plants of interest include cauliflower (Brassica oleracea), artichoke(Cynara scolvmus), and safflower (Carthamus, e.g. tinctorius); fruitssuch as apple (Malus, e.g. domesticus), banana (Musa, e.g. acuminata),berries (such as the currant, Ribes, e.g. rubrum), cherries (such as thesweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus),grape (Vitis, e.g. vinifera), lemon (Citrus limon), melon (Cucumismelo), nuts (such as the walnut, Juglans, e.g. regia; peanut, Arachishypoaeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica),pear (Pyra, e.g. communis), pepper (Solanum, e.g. capsicum), plum(Prunus, e.g. domestica), strawberry (Fragaria, e.g. moschata), tomato(Lycopersicon, e.g. esculentum); leafs, such as alfalfa (Medicago, e.g.sativa), sugar cane (Saccharum), cabbages (such as Brassica oleracea),endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce(Lactuca, e.g. sativa), spinach (Spinacia e.g. oleraceae), tobacco(Nicotiana, e.g. tabacum); roots, such as arrowroot (Maranta, e.g.arundinacea), beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota),cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g. rapa), radish(Raphanus, e.g. sativus) yam (Dioscorea, e.g. esculenta), sweet potato(Ipomoea batatas); seeds, such as bean (Phaseolus, e.g. vulgaris), pea(Pisum, e.g. sativum), soybean (Glycine, e.g. max), wheat (Triticum,e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. mays),rice (Oryza, e.g. sativa); grasses, such as Miscanthus grass(Miscanthus, e.g., giganteus) and switchgrass (Panicum, e.g. virgatum);trees such as poplar (Populus, e.g. tremula), pine (Pinus); shrubs, suchas cotton (e.g., Gossypium hirsutum); and tubers, such as kohlrabi(Brassica, e.g. oleraceae), potato (Solanum, e.g. tuberosum), and thelike.

Plant Transformation

The expression constructs described herein can be introduced into theplant cell in a number of art-recognized ways. The term “introducing” inthe context of a polynucleotide, for example, a nucleotide construct ofinterest, is intended to mean presenting to the plant the polynucleotidein such a manner that the polynucleotide gains access to the interior ofa cell of the plant. Where more than one polynucleotide is to beintroduced, these polynucleotides can be assembled as part of a singlenucleotide construct, or as separate nucleotide constructs, and can belocated on the same or different transformation vectors. Accordingly,these polynucleotides can be introduced into the host cell of interestin a single transformation event, in separate transformation events, or,for example, in plants, as part of a breeding protocol. The methods ofthe invention do not depend on a particular method for introducing oneor more polynucleotides into a plant, only that the polynucleotide(s)gains access to the interior of at least one cell of the plant. Methodsfor introducing polynucleotides into plants are known in the artincluding, but not limited to, transient transformation methods, stabletransformation methods, and virus-mediated methods.

“Transient transformation” in the context of a polynucleotide isintended to mean that a polynucleotide is introduced into the plant anddoes not integrate into the genome of the plant.

By “stably introducing” or “stably introduced” in the context of apolynucleotide introduced into a plant is intended the introducedpolynucleotide is stably incorporated into the plant genome, and thusthe plant is stably transformed with the polynucleotide.

“Stable transformation” or “stably transformed” is intended to mean thata polynucleotide, for example, a nucleotide construct described herein,introduced into a plant integrates into the genome of the plant and iscapable of being inherited by the progeny thereof, more particularly, bythe progeny of multiple successive generations.

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation arts, andthe genes pertinent to this invention can be used in conjunction withany such vectors. The selection of vector will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptll gene, which confers resistance tokanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268(1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, whichconfers resistance to the herbicide phosphinothricin (White et al.,Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79:625-631 (1990)), the hph gene, which confers resistance to theantibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4:2929-2931), and the dhfr gene, which confers resistance to methatrexate(Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, whichconfers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and5,188,642), and the mannose-6-phosphate isomerase gene, which providesthe ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and5,994,629).

Methods for regeneration of plants are also well known in the art. Forexample, Ti plasmid vectors have been utilized for the delivery offoreign DNA, as well as direct DNA uptake, liposomes, electroporation,microinjection, and microprojectiles. In addition, bacteria from thegenus Agrobacterium can be utilized to transform plant cells. Below aredescriptions of representative techniques for transforming bothdicotyledonous and monocotyledonous plants, as well as a representativeplastid transformation technique.

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Forthe construction of vectors useful in Agrobacterium transformation, see,for example, US Patent Application Publication No. 2006/0260011, hereinincorporated by reference.

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vectorand consequently vectors lacking these sequences can be utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. Transformation techniques that do not rely on Agrobacteriuminclude transformation via particle bombardment, protoplast uptake (e.g.PEG and electroporation) and microinjection. The choice of vectordepends largely on the preferred selection for the species beingtransformed. For the construction of such vectors, see, for example, USApplication No. 20060260011, herein incorporated by reference.

For expression of a nucleotide sequence of the present invention inplant plastids, plastid transformation vector pPH143 (WO 97/32011,example 36) is used. The nucleotide sequence is inserted into pPH143thereby replacing the PROTOX coding sequence. This vector is then usedfor plastid transformation and selection of transformants forspectinomycin resistance. Alternatively, the nucleotide sequence isinserted in pPH143 so that it replaces the aadH gene. In this case,transformants are selected for resistance to PROTOX inhibitors.

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., EMBO J. 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth of these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Frommet al. (Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200(1993)) describe techniques for the transformation of elite inbred linesof maize by particle bombardment. This technique utilizes immature maizeembryos of 1.5-2.5 mm length excised from a maize ear 14-15 days afterpollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation.

Patent Application EP 0 332 581 describes techniques for the generation,transformation and regeneration of Pooideae protoplasts. Thesetechniques allow the transformation of Dactylis and wheat. Furthermore,wheat transformation has been described by Vasil et al. (Biotechnology10: 667-674 (1992)) using particle bombardment into cells of type Clong-term regenerable callus, and also by Vasil et al. (Biotechnology11:1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084(1993)) using particle bombardment of immature embryos and immatureembryo-derived callus. A preferred technique for wheat transformation,however, involves the transformation of wheat by particle bombardment ofimmature embryos and includes either a high sucrose or a high maltosestep prior to gene delivery. Prior to bombardment, any number of embryos(0.75-1 mm in length) are plated onto MS medium with 3% sucrose(Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l2,4-D for induction of somatic embryos, which is allowed to proceed inthe dark. On the chosen day of bombardment, embryos are removed from theinduction medium and placed onto the osmoticum (i.e. induction mediumwith sucrose or maltose added at the desired concentration, typically15%). The embryos are allowed to plasmolyze for 2-3 hours and are thenbombarded. Twenty embryos per target plate is typical, although notcritical. An appropriate gene-carrying plasmid (such as pCIB3064 orpSOG35) is precipitated onto micrometer size gold particles usingstandard procedures. Each plate of embryos is shot with the DuPontBIOLISTICS® helium device using a burst pressure of about 1000 psi usinga standard 80 mesh screen. After bombardment, the embryos are placedback into the dark to recover for about 24 hours (still on osmoticum).After 24 hrs, the embryos are removed from the osmoticum and placed backonto induction medium where they stay for about a month beforeregeneration. Approximately one month later the embryo explants withdeveloping embryogenic callus are transferred to regeneration medium(MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriateselection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/lmethotrexate in the case of pSOG35). After approximately one month,developed shoots are transferred to larger sterile containers known as“GA7s” which contain half-strength MS, 2% sucrose, and the sameconcentration of selection agent.

Transformation of monocotyledons using Agrobacterium has also beendescribed. See, WO 94/00977 and U.S. Pat. No. 5,591,616, and, Negrottoet al., Plant Cell Reports 19: 798-803 (2000).

For example, rice (Oryza sativa) can be used for generating transgenicplants. Various rice cultivars can be used (Hiei et al., 1994, PlantJournal 6:271-282; Dong et al., 1996, Molecular Breeding 2:267-276; Hieiet al., 1997, Plant Molecular Biology, 35:205-218). Also, the variousmedia constituents described below may be either varied in quantity orsubstituted. Embryogenic responses are initiated and/or cultures areestablished from mature embryos by culturing on MS-CIM medium (MS basalsalts, 4.3 g/liter; B5 vitamins (200×), 5 ml/liter; Sucrose, 30 g/liter;proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300mg/liter; 2,4-D (1 mg/ml), 2 ml/liter; adjust pH to 5.8 with 1 N KOH;Phytagel, 3 g/liter). Either mature embryos at the initial stages ofculture response or established culture lines are inoculated andco-cultivated with the Agrobacterium tumefaciens strain LBA4404(Agrobacterium) containing the desired vector construction.Agrobacterium is cultured from glycerol stocks on solid YPC medium (100mg/L spectinomycin and any other appropriate antibiotic) for about 2days at 28° C. Agrobacterium is re-suspended in liquid MS-CIM medium.The Agrobacterium culture is diluted to an OD600 of 0.2-0.3 andacetosyringone is added to a final concentration of 200 uM.Acetosyringone is added before mixing the solution with the ricecultures to induce Agrobacterium for DNA transfer to the plant cells.For inoculation, the plant cultures are immersed in the bacterialsuspension. The liquid bacterial suspension is removed and theinoculated cultures are placed on co-cultivation medium and incubated at22° C. for two days. The cultures are then transferred to MS-CIM mediumwith Ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium.For constructs utilizing the PMI selectable marker gene (Reed et al., InVitro Cell. Dev. Biol.-Plant 37:127-132), cultures are transferred toselection medium containing Mannose as a carbohydrate source (MS with 2%Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3-4weeks in the dark. Resistant colonies are then transferred toregeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1mg/liter zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) andgrown in the dark for 14 days. Proliferating colonies are thentransferred to another round of regeneration induction media and movedto the light growth room. Regenerated shoots are transferred to GA7containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2weeks and then moved to the greenhouse when they are large enough andhave adequate roots. Plants are transplanted to soil in the greenhouse(T0 generation) grown to maturity, and the T1 seed is harvested.

The plants obtained via transformation with a nucleic acid sequence ofthe present invention can be any of a wide variety of plant species,including those of monocots and dicots; however, the plants used in themethod of the invention are preferably selected from the list ofagronomically important target crops set forth supra. The expression ofa gene of the present invention in combination with othercharacteristics important for production and quality can be incorporatedinto plant lines through breeding. Breeding approaches and techniquesare known in the art. See, for example, Welsh J. R., Fundamentals ofPlant Genetics and Breeding, John Wiley & Sons, NY (1981); CropBreeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis.(1983); Mayo O., The Theory of Plant Breeding, Second Edition, ClarendonPress, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseasesand Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding, Walter de Gruyterand Co., Berlin (1986).

For the transformation of plastids, seeds of Nicotiana tabacum c.v.“Xanthiene” are germinated seven per plate in a 1″ circular array on Tagar medium and bombarded 12-14 days after sowing with 1 um tungstenparticles (M10, Biorad, Hercules, Calif.) coated with DNA from plasmidspPH143 and pPH145 essentially as described (Svab, Z. and Maliga, P.(1993) PNAS 90, 913-917). Bombarded seedlings are incubated on T mediumfor two days after which leaves are excised and placed abaxial side upin bright light (350-500 umol photons/m²/s) on plates of RMOP medium(Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530)containing 500 μg/ml spectinomycin dihydrochloride (Sigma, St. Louis,Mo.). Resistant shoots appearing underneath the bleached leaves three toeight weeks after bombardment are subcloned onto the same selectivemedium, allowed to form callus, and secondary shoots isolated andsubcloned. Complete segregation of transformed plastid genome copies(homoplasmicity) in independent subclones is assessed by standardtechniques of Southern blotting (Sambrook et al., (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor). BamHI/EcoRI-digested total cellular DNA (Mettler, I. J. (1987)Plant Mol Biol Reporter 5, 346349) is separated on 1% Tris-borate (TBE)agarose gels, transferred to nylon membranes (Amersham) and probed with.sup.32P-labeled random primed DNA sequences corresponding to a 0.7 kbBamHI/HindIII DNA fragment from pC8 containing a portion of the rps7/12plastid transit sequence. Homoplasmic shoots are rooted asepticallyon spectinomycin-containing MS/IBA medium (McBride, K. E. et al. (1994)PNAS 91, 7301-7305) and transferred to the greenhouse.

The genetic properties engineered into the transgenic seeds and plantsdescribed above are passed on by sexual reproduction or vegetativegrowth and can thus be maintained and propagated in progeny plants.Generally, maintenance and propagation make use of known agriculturalmethods developed to fit specific purposes such as tilling, sowing orharvesting.

Use of the advantageous genetic properties of the transgenic plants andseeds according to the invention can further be made in plant breeding.Depending on the desired properties, different breeding measures aretaken. The relevant techniques are well known in the art and include butare not limited to hybridization, inbreeding, backcross breeding,multi-line breeding, variety blend, interspecific hybridization,aneuploid techniques, etc. Thus, the transgenic seeds and plantsaccording to the invention can be used for the breeding of improvedplant lines that, for example, increase the effectiveness ofconventional methods such as herbicide or pesticide treatment or allowone to dispense with said methods due to their modified geneticproperties.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by J. Sambrook, E. F. Fritschand T. Maniatis, Molecular Cloning: A Laboratory manual, Cold SpringHarbor laboratory, Cold Spring Harbor, N.Y. (1989) and by T. J. Silhavy,M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and byAusubel, F. M. et al., Current Protocols in Molecular Biology, pub. byGreene Publishing Assoc. and Wiley-Interscience (1987).

Example 1 Biochemical Method of Identifying a Chloroplast TransitPeptide Sequence from a Glaucocystophyte

A Glaucocystophyte will be grown essentially as described by Bayer andSchenk, Curr Genet 16: 311-313 (1989). The culture will be grown withcontinuous light at 24 degrees C. and 4% carbon dioxide. The cyanelleorganelle will be isolated essentially as described by Bayer and Schenk,Curr Genet 16: 311-313 (1989) by first harvesting the cells bycentrifugation followed by organelle isolation by osmotic shock (Zookand Schenk, Endocyt Cell Res 3: 203-211 (1986)). The isolated cyanellesare further treated with DNasI and subsequently washed in phosphatebuffered saline containing 1 mM magnesium chloride and 1 mM calciumchloride.

Cyanelle proteins will be recovered by lysing the purified cyanelleswith lysozyme (0.5 micrograms per microliter of the above phosphatebuffered saline solution). The cyanelle proteins will be purified fromthe lysate by desalting in water and concentrating the resulting lysateusing standard techniques.

The cyanelle proteins will be end sequenced to derive an amino terminalpartial sequence of the proteins. The protein sequence will be comparedto a database containing genomic DNA sequence to identify candidate openreading frames in the nuclear DNA that encodes the cyanelle targetedprotein. The cyanelle transit peptide sequence is derived by comparingthe amino terminal sequence of isolated protein and the coding region ofthe nuclear gene to identify the amino terminal extension which servesas the cyanelle transit peptide sequence.

Example 2 Plant Transformation

2.A. Maize Transformation:

Transformation of immature maize embryos is performed essentially asdescribed in Negrotto et al., Plant Cell Reports 19:798-803 (2000).Various media constituents described therein can be substituted.

Agrobacterium strain LBA4404 (Invitrogen) containing the planttransformation plasmid is grown on YEP (yeast extract (5 g/L), peptone(10 g/L), NaCl (5 g/L), 15 g/l agar, pH 6.8) solid medium for 2 to 4days at 28° C. Approximately 0.8×10⁹ Agrobacteria are suspended inLS-inf media supplemented with 100 μM acetosyringone (As) (LSAs medium)(Negrotto et al., Plant Cell Rep 19:798-803 (2000)). Bacteria arepre-induced in this medium for 30-60 minutes.

Immature embryos from maize line, A188, or other suitable maizegenotypes are excised from 8-12 day old ears into liquid LS-inf+100 μMAs (LSAs). Embryos are vortexed for 5 seconds and rinsed once with freshinfection medium. Infection media is removed and Agrobacterium solutionis then added and embryos are vortexed for 30 seconds and allowed tosettle with the bacteria for 5 minutes. The embryos are then transferredscutellum side up to LSAs medium and cultured in the dark for two tothree days. Subsequently, between 20 and 25 embryos per petri plate aretransferred to LSDc medium supplemented with cefotaxime (250 mg/l) andsilver nitrate (1.6 mg/l) (Negrotto et al., Plant Cell Rep 19:798-803(2000)) and cultured in the dark for 28° C. for 10 days.

Immature embryos producing embryogenic callus are transferred toLSD1M0.5S medium (LSDc with 0.5 mg/l 2,4-D instead of Dicamba, 10 g/lmannose, 5 g/l sucrose and no silver nitrate). The cultures are selectedon this medium for 6 weeks with a subculture step at 3 weeks. Survivingcalli are transferred either to LSD1M0.5S medium to be bulked-up or toReg1 medium (as described in Negrotto et al., Plant Cell Rep 19:798-803(2000). Following culturing in the light (16 hour light/8 hour darkregiment), green tissues are then transferred to Reg2 medium withoutgrowth regulators (as described in Negrotto et al., Plant Cell Rep19:798-803 (2000) and incubated for 1-2 weeks. Plantlets are transferredto Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3medium (as described in Negrotto et al. 2000) and grown in the light.Plants that are PCR positive for any component of the expressioncassette are transferred to soil and grown in the greenhouse.

The presence of any component of the expression cassette is determinedby +/−PCR assay or by a Taqman copy number assay.

2.B. Tobacco Transformation:

Agrobacterium tumefaciens strain LBA4404 containing a transformationvector containing an expression cassette are used to infect leafexplants of Nicotiana tabacum c.v. Petit Havana (SR1). Initially, thetobacco leaves are cut into 1-2 mm wide slices, exposed to theAgrobacterium for 5 minutes, and are placed on sterile paper to blotaway excess liquid and then placed on co-cultivation medium for 3 days.The leaf slices are moved to selection/regeneration medium containingthe appropriate selection agent. Selection agent resistant shoots aretransplanted to soil. The plants are selfed or outcrossed with pollensfrom nontransgenic SR1 plants to produce seeds. The presence of anycomponent of the expression cassette is determined by +/−PCR assay or bya Taqman copy number assay.

Example 3 Cyanophora paradoxa FNR Chloroplast Transit Peptide Sequenceand cZsGreen in Maize

A cloning vector was created whereby the FNR-transit peptide was fusedin-frame to a reporter gene (cZsGreen) with expression driven by theCestrum Yellow Leaf Curl viral promoter and the NOS-gene terminator.This expression cassette was ligated into a plant transformation binaryvector and subsequently used to generate Agrobacterium mediatedtransgenic maize events essentially as described in Example 2.

Western blot analysis of the T0-maize events found the reporter gene isexpressed and that the reporter gene protein was cleaved to a maturesize. Subsequently, chloroplasts were isolated from green leaf tissuesfrom both transgenic and non-transgenic maize plants. Fluorescencemicroscopy (λ=496 nm excitation, λ=506 nm emission) revealedauto-fluorescence in chloroplasts isolated from the transgenic eventsbut not from the negative control non-transgenic chloroplasts confirmingthe targeting of the expressed reporter gene protein to the chloroplast.

Example 4 Cyanophora paradoxa FNR Chloroplast Transit Peptide Sequenceand Endoglucanase in Maize and Tobacco

4.A: Expression Cassette Construction

Expression vectors capable of directing the expression of endoglucanasein transgenic plants were designed for both monocot and dicot optimizedendoglucanase. Tobacco expression vectors used the constitutive promoterCestrum yellow leaf curl virus (CYLCV) promoter to drive expression ofthe dicot optimized endoglucanase gene. Plastid targeting of theendoglucanase was via the transit peptide (SEQ ID NO:2) fromferredoxin-NADP+reductase (FNR) of Cyanophora paradoxa fused to theN-terminus (FEBS Letters 1996: 381, 153-155).

The maize PepC promoter (The Plant Journal 1994: 6(3), 311-319) was usedto drive maize leaf specific expression of the monocot optimizedendoglucanase. Plastid targeted constructs contained the FNR transitpeptide as described in Example 3. All expression cassettes weresubcloned into a binary vector for transformation into tobacco and maizeusing recombinant DNA techniques that are known in the art.

4.B: Transgenic Maize Plants Characterization

Protein extracts were obtained from approximately 100-500 mg of leaftissue or 100 mg flour generated from maize seed from non-transgenic andtransgenic plants. Leaf material was placed into 96 deep well blockscontaining small steel balls and pre-cooled on dry ice. Samples wereground to a fine powder using a Geno/Grinder (SPEC/CertiPrep, Metuchen,N.J.). Flour samples were prepared by pooling approximately 10-20 seedand grinding to a fine powder using a KLECO Grinder (Gracia MachineCompany, Visalia, Calif.).

Preparation of samples for SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and western blot analysis was carried out by extracting 50 to100 mg leaf material in 250-500 μl of either Western Extraction Buffer(WEB=12.5 mM sodium borate, pH 10; 2% BME; and 1% SDS) or assay buffer,as described below, at room temperature for approximately 30 minutesfollowed by centrifugation for 5 minutes at 13,000 rpm.

SDS-PAGE was performed by transferring 100 μl of WEB samples to aneppendorf tube and add 25 μl 4XBioRad LDS or modified BioRad loadingbuffer (4×BioRad LDS:BME at a ratio of 2:1). Samples were heated for 10minutes at 70° C. then immediately place on ice for 5 minutes. Spinsamples briefly, and transfer back on to ice. Sample extracts (5-10 μl)were run on BioRad 4-12% Bis/Tris protein gel (18 well) using MOPSbuffer.

Immunoblot analysis was performed by transferring SDS-PAGE gels onto anitrocellulose membrane using chilled Nupage transfer buffer(Invitrogen) for 30 minutes at 100 volts. Total protein transferred tothe blot was visualized using Ponceau stain (Sigma). Following Ponceaustaining, the membrane was incubated in blocking buffer for 30 minutesin TBST wash buffer (30 mM Tris-HCL, pH 7.5, 100 mM NaCl, and 0.05%Tween 20) with 3% dry milk, then washed three times for 5 minutes inTBST. Polyclonal goat or rabbit primary antibody was added at 1 ug/ml inTBST wash buffer with 3% milk, and the blot incubated 2 hours toovernight. Following overnight incubation, the blot was washed threetimes for 5 minutes each in TBST wash buffer. Secondary antibody(Rabbit-AP or Goat-AP) was diluted 1:8000 (in TBST) and added to blotfor 30 minutes. Following incubation in the secondary antibody, the blotwas again washed three times for 5 minutes each. Visualization of immunoreactive bands was carried out by adding Moss BCIP/NBT-alkalinephosphatase substrate. Blots were rinsed thoroughly in water followingincubation in the BCIP/NBT substrate and allowed to air dry.

Approximately 100-500 mg of fresh leaf tissue of approximately 15 dayold T0 transgenic plant was extracted in 2 to 10 ml of one of thefollowing buffers: (A) 100 mM Na acetate, 0.02% Tween, 0.02% Na azide pH4.75, 1% PVP and Complete protease inhibitor cocktail tablets (Roche) or(B) 100 mM Na acetate, 0.02% Tween, and 0.02% Na azide pH 4.75 andComplete protease inhibitor cocktail tablets (Roche); or (C) 1 mM 0.02%Tween-20, 0.02% NaN₃ Alternative buffers for extracting protein fromleaf are well known in the art. Samples were placed on benchtop rotatorsfor 30-60 minutes then centrifuged at 3000 rpm for 10 minutes. For freshleaf samples, the amount of total protein extracted was measured byPierce BCA protocol as outlined in product literature. Endoglucanaseactivity assays were carried out using one of the following substrates:pNP-lactoside, methylumbelliferyl-lactoside (MUL),carboxymethyl-cellulose (CMC), oat-μ glucan, phosphoric acid treatedcellulose (PASC), Avicel, or other available substrates. Othersubstrates can be used for measuring cellulase activity followingpreviously published protocols (Methods in Enzymology, Vol 160).

The following describes the method used for extracting and assaying,transgenically-expressed endoglucanase from leaf tissue. This methodmeasures endoglucanase enzyme in μmol/min/mg of protein in terms ofliberated glucose produced on CM-cellulose (0.5%) at 40° C., pH 4.75.Glucose oxidase/peroxidase (GOPOD) chemistry is used in this assay tomeasure glucose relative to a standard curve. The glucose-based assaymethod is a colorimetric assay in which GOPOD reacts with glucose at 40°C. to generate a light to dark pinkish chromophore. The assay consistsof 4 basic steps: (1) grinding/milling transgenic tissue, (2) weighingout ground tissue samples, (3) extracting enzyme in Na Acetate buffer,and (4) assaying for enzymatic activity/protein quantification.

Enzymatic data and western blot data for transgenic maize plantsexpressing endoglucanase is outlined in Table 2. Table 3 outlinestransgenic tobacco data generated as described above. The enzymatic datagenerated in table 2 and 3 was performed using CMC as the substrate asdescribed above.

TABLE 2 T0 transgenic maize plants expressing endoglucanase operablylinked to the FNR chloroplast transit peptide sequence from Cyanophoraparadoxa. Activity Transgenic line nmol/min/mg Standard Western numberprotein deviation blot 002A 14.02 0.70 positive 003A 10.19 0.59 positive004A 15.09 0.85 positive 006A 10.13 0.49 positive 007A 17.53 0.66positive 008A 16.08 0.62 positive 011A 9.95 0.42 positive 015A 16.980.88 positive 016A 18.15 1.31 positive Non-transgenic 0.40 0.09 negativenegative control 021A 13.67 0.54 positive 023A 14.20 0.38 positive 024A9.72 0.36 positive 025A 12.99 0.53 positive 027A 15.05 0.66 positive030A 17.27 0.64 positive 033A 14.08 0.62 positive 034A 10.36 0.79positive 045A 12.54 0.81 positive 047A 12.14 0.67 positive 050A 12.750.76 positive 051A 18.21 1.12 n.d. 055A 11.07 0.58 n.d. 060A 9.69 0.41n.d. 068A 11.77 0.18 n.d. 075A 19.67 1.02 n.d. 076A 8.49 0.59 n.d. 081A9.72 0.65 n.d. 084A 11.52 0.69 n.d. 085A 9.21 0.62 n.d. 086A 11.12 0.76n.d. 087A 13.89 1.17 n.d. 089A 13.15 2.04 n.d. 098A 15.01 1.00 n.d. 101A12.56 0.70 n.d. 105A 6.94 0.55 n.d. 106A 11.07 0.42 n.d. 108A 11.61 0.41n.d. 115A 7.62 0.31 n.d. 120A 12.02 0.73 n.d. 121A 9.93 8.04 n.d. 125A12.51 0.74 n.d. 127A 8.97 3.16 n.d. 128A 17.94 2.07 n.d. 133A 12.15 9.85n.d. 134A 8.98 1.26 n.d. 135A 12.46 0.13 n.d. n.d. = not determined

TABLE 3 TO Transgenic tobacco endoglucanase activity data forendoglucanase operably linked to the FNR chloroplast transit peptidesequence from Cyanophora paradoxa. Transgenic tobacco line Avgnmol/min/mg TSP STDev T001A 12.91 0.33 T004A 14.45 5.47 T007A 5.54 0.24T011A 5.42 0.73 T013B 19.80 0.72 T017A 3.82 0.09 T018A 8.61 0.54 T019Anegative −1.01 1.82 control ? T021A 0.54 4.50 T023B 11.83 0.61Non-transgenic 0.78 0.28

4.C: Chloroplast Targeting

The above described transgenic maize and tobacco plants will be used toisolate chloroplasts using the Chloroplast Isolation Kit (Sigma)following the manufacturers directions. The isolated chloroplasts willbe treated with the peptidase, thermolysin, to remove proteins bound tothe outer membrane of the isolated chloroplast. Total protein will beisolated from the treated chloroplasts using standard techniques and theendoglucanase activity measured essentially as described in Example 4.

Example 5 Additional Chloroplast Transit Peptide Sequences Derived fromCyanophora paradoxa

Additional cyanelle transit peptides will be evaluated in transgeniccorn plants. The cyanelle transit peptides Rieske1 (transit peptide ofSEQ ID NO:3), Rieske2 (maize optimized coding sequence of SEQ ID NO:4and transit peptide sequence of SEQ ID NO:5) and cytochrome c6 (maizeoptimized coding sequence of SEQ ID NO:6 and transit peptide sequence ofSEQ ID NO:7) from Cyanophora paradoxa will be cloned to the 5′ end of areporter gene such that the cyanelle transit peptide is at theamino-terminus of the encoded polypeptide of a reporter gene. Thereporter gene could be a selectable marker, GUS, CzsGreen or AmCyan (SEQID NO:8). The polynucleotide sequences of SEQ ID NOs: 6 and 8 will becloned to the 5′ end of a selectable marker, GUS, CzsGreen or AmCyansuch that the transit peptide is at the amino-terminus of the encodedpolypeptide. The reporter gene could be a selectable marker, GUS,CzsGreen or AmCyan.

The coding sequence described above will be cloned into an expressioncassette such that the coding sequence is operably linked to a cestrumpromoter. The expression cassette will be cloned into a transformationvector containing the PPO gene as a selectable marker for planttransformation. Transgenic maize or tobacco plants will be generatedusing standard transformation techniques. Transgenic events will beconfirmed by Taqman analysis designed to detect any of the componentswithin the expression cassette.

TABLE 4 Cyanophora paradoxa cyanelle transit sequences. SEQ ID NO: Genename 1 Ferredoxin NADP oxidoreductase transit peptide, maize optimizedcoding sequence 2 Ferredoxin NADP oxidoreductase transit peptidesequence 3 Rieske1 transit peptide 4 Rieske2 transit peptide, maizeoptimized coding sequence 5 Rieske2 transit peptide 6 Cytochrome c6transit peptide, maize optimized coding sequence 7 Cytochrome c6 transitpeptide

The transit peptides will be evaluated using both western blots(immuno-blotting) and fluorescence. Whole plant extracts from transgenicplant leaf tissue will be prepared as described in the followingparagraph and run on an 8-16% tris-glycine gel. The gel will be blottedand then probed with an antibody to the reporter gene (i.e. antibody toAmCyan). Two bands are expected corresponding to the processed (AmCyanwithout transit peptide) and unprocessed (transit peptide and AmCyan)form of the protein. From there, chloroplast preparations will be madeby isolating chloroplasts from the transgenic leaf tissue using theChloroplast Isolation Kit (Sigma). The purified chloroplasts will belysed and the resulting lysate run on an 8-16% tris-glycine gel, blottedand probed with the antibody to the reporter gene (such as theanti-AmCyan antibody). We expect to find a band whose size correspondsto the processed form of the protein and the absence of a band thatwould correspond to an unprocessed form of the protein.

Western blots from whole extracts are performed by placing 3 holepunches of leaf tissue into a microcentrifuge tube and adding 100 μl ofLaemmli buffer. The punches are then ground using a pestle and thepreparation is then boiled at 100° C. for 10 min. The sample is thencentrifuged at 13000 rpm (Eppendorf Centrifuge 5417R) and 10 ml of thesupernatant are loaded onto a gel (8-16% tris-glycine, Invitrogen). Thegel is run at 150V for 60 min. The separated proteins are thentransferred to a PVDF membrane via electrophoresis (350 mA for 60 min.).The membrane is blocked for 1 hour at 25° C. in 5% milk. Blockedmembranes were placed in a primary antibody solution of 1:5000 in PBST(PBS with 0.1% TWEEN 20) and placed on a rocker at 25° C. for 2 hours.The membrane is removed from the primary solution, washed in PBST (5, 10min washes on a rocker at 25° C.) and placed in a secondary antibodysolution (1:5000 dilution in PBST) for 2 hours at 25° C. on a rocker.The membrane is washed (5, 10 min washes on a rocker at 25° C.) anddeveloped in BCIP. The BCIP reaction is stopped with tap water and thedeveloped blot is allowed to dry at room temperature.

When using the AmCyan as a reporter gene, we can use confocal microscopy(Zeiss, LSM 710) to analyze the reporter protein (AmCyan) in relation toendogenous, auto-fluorescence within the cell. For our experiments wewould expect the AmCyan fluorescence and the chlorophyllauto-fluorescence to co-localize within the cell when the transitpeptide is performing as expected.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid comprising a first nucleotide sequenceencoding a plastid transit peptide from a Glaucocystophyte wherein saidfirst nucleotide sequence is operably linked to a second nucleotidesequence encoding a heterologous polypeptide.
 2. The isolated nucleicacid of claim 1, wherein the Glaucocystophyte is Cyanophora.
 3. Theisolated nucleic acid of claim 1, wherein the Glaucocystophyte isCyanophora paradoxa.
 4. The isolated nucleic acid of claim 1, whereinthe plastid transit peptide is selected from the group consisting of SEQID NOs: 2, 3, 5 and
 7. 5. A transgenic plant comprising a firstnucleotide sequence encoding a plastid transit peptide from aGlaucocystophyte wherein said first nucleotide sequence is operablylinked to a second nucleotide sequence encoding a heterologouspolypeptide, wherein said heterologous polypeptide is targeted to achloroplast.
 6. The transgenic plant of claim 5, wherein theGlaucocystophyte is Cyanophora.
 7. The transgenic plant of claim 5,wherein the Glaucocystophyte is Cyanophora paradoxa.
 8. The transgenicplant of claim 5, wherein the plant is a monocot.
 9. The transgenicplant of claim 5, wherein the plant is a dicot.
 10. The transgenic plantof claim 5, wherein the plastid transit peptide encodes the polypeptideselected from the group consisting of SEQ ID NOs:2, 3, 5 and
 7. 11. Thetransgenic plant of claim 8, wherein the monocot is a maize or asugarcane plant.
 12. The transgenic plant of claim 9, wherein the dicotis a soybean or a sugarbeet plant.
 13. A method of stably expressing aheterologous polypeptide in a plant, said method comprising introducinginto said plant a DNA construct comprising a first nucleotide sequenceencoding a plastid transit peptide from a Glaucocystophyte, wherein saidfirst nucleotide sequence is operably linked to a second nucleotidesequence encoding said heterologous polypeptide.
 14. The method of claim13, wherein the Glaucocystophyte is Cyanophora.
 15. The method of claim13, wherein the Glaucocystophyte is Cyanophora paradoxa.
 16. The methodof claim 13, wherein the plant is a monocot.
 17. The method of claim 13,wherein the plant is a dicot.
 18. The method of claim 13, wherein theplastid transit peptide encodes the polypeptide selected from the groupconsisting of SEQ ID NO:2, 3, 5 and
 7. 19. The method of claim 16,wherein the monocot is a maize or a sugarcane plant.
 20. The method ofclaim 17, wherein the dicot is a soybean or a sugarbeet plant.
 21. Anisolated polypeptide comprising a plastid transit peptide from aGlaucocystophyte operably linked to a heterologous polypeptide.
 22. Theisolated polypeptide of claim 21, wherein the Glaucocystophyte isCyanophora.
 23. The isolated polypeptide of claim 21, wherein theGlaucocystophyte is Cyanophora paradoxa.
 24. The isolated polypeptide ofclaim 21, wherein the plant is a monocot.
 25. The isolated polypeptideof claim 21, wherein the plant is a dicot.
 26. The isolated polypeptideof claim 21, wherein the plastid transit sequence encodes thepolypeptide selected from the group consisting of SEQ ID NOs:2, 3, 5,and
 7. 27. The isolated polypeptide of claim 24, wherein the monocot isa maize or a sugarcane plant.
 28. The isolated polypeptide of claim 25,wherein the dicot is a soybean or a sugarbeet plant.